Week 10

08/03 - 08/07

Toxicity and solubility of TPA

1. Dissolution of TPA in DMSO (best concentration of 62mg/L)
2. Dilution of TPA stock in LB Broth => TPA precipitates at a concentration of 3,9g/L, maximum concentration in LB Broth is 0,9 mg/L

Preparation of M9 and minimal media
3 solutions:
-
Solution 1: (salts)
5,3g of Potassium Phosphate (dibasic) K2HPO4
2g of Potassium Phosphate (monobasic) KH2PO4
1g of Ammonium Sulfate (NH4)2SO4.
0,5g of Sodium Citrate (tribasic, dihydrate)
Autoclave
Complete with water to 333 mL

Solution 2: (agar)
16g of agar
Complete with water to 333 mL
Autoclave

Solution 3: (sugar)
In fact we have to put 4g of sugar in 333 mL of H2O. But we wanted to see if bacteria took TPA for a carbon source. So just the water was put. Next the flacon was autoclaved.

When the three parts have been autoclaved, 2 ingredients must be add to the medium in aseptically conditions:
1mL of 10% Magnesium Sulfate MgSO4 (autoclaved too) (1,5g in 15mL of H2O).
1mL of 0,2% Thiamin (vitamin B1).

At the end the volume of Agar was too important (350mL and not 333mL). So we had to redo this experience the next day.

05-08-15 – Experiment: Preparation of M9 medium – VB, PV
Redaction by VB

Aim: Get medium for Pfeifer cells culture.

Protocol: (for 1L medium)

Agar was directly put in a bottle and salts were dissolved in a beaker with water.

6g of Sodium Phosphate Na2HPO4.H2O
3g of Potassium Phosphate KH2PO4
0,5g of Sodium chloride NaCl
1g of Ammonium chloride NH4Cl
16g of agar
Complete with water to 1L

When this flacon was autoclaved some ingredients were added in aseptically conditions:

1mL of 1M Magnesium sulphate MgSO4 (6,02g in 50mL H2O)
1mL of 0,1M of Calcium Chloride CaCl2 (0,55g in 50mL of H2O)

A mistake was done yesterday but this time an other did too: The last ingredients were added but they were not sterile so the medium failed again.

06-08-2015 – Experiment: Minimal and M9 medium for TPA Toxicity and BAP – VB, PV
Redaction by VB

Aim: Have prepared M9 Medium and Minimal Medium for Petri dishes

We restart the two last protocols but this time we included the agar for the future Petri dishes.
For the future experiments every medium will have the same concentration of sugar for the bacteria growth, and the test of the TPA toxicity.  We began by a theoretical concentration for our Petri dishes: to see the saturation concentration of TPA we decided to do dilutions at 1/10 and 1/20.
This time we decided to make 1,5L of Minimal Medium
Protocol:

Solution 1: (salts)
8g of Potassium Phosphate K2HPO4
3g of Potassium Phosphate KH2PO4
1,5g of Ammonium Sulfate (NH4)2SO4
0,75g of Sodium Citrate (tribasic, dihydrate) Na3C6H5O7.(H2O) 2
Complete with water to 1L

Solution 2: (agar)
24g of Agar
Complete with water to 500 mL

Solution 3:
500 mL of H2O

We underestimate the time for agar to solidify, so we had solid agar in an eprouvette. Moreover one of the flacon was not sterile so we contaminated our medium. We decided to return at the first protocol (for 1L).

At the end of this journey the two mediums were ready but they did not have the last ingredients.

Enzymatic Assay of NB-Esterase

1. XbaI ans PstI enzymatic digest of BBa_K808030 and pDG011
2. Gel Purification of the digested fragments on 0,8% Agarose Gel.
3. Ligation of pDG011 and BBa_K808030 using T4 DNA ligase.
4. Gel Migration on 0,8% Agarose Gel.

Gibson Assembly

1. Ligation of ThpA1, Transp_, 1392932_, using T4 DNA ligase.
2. DNA concentration measurement using a spectrophotometer.